The therapeutic efficacy of pharmaceutical or therapeutic agents relies on the delivery of adequate doses of a pharmaceutical agent to the site of action. Many modes of delivery have been developed, including, for example, enteral (oral), parenteral (intramuscular, intravenous, subcutaneous), and topical administration. In most instances the administration system is chosen for reliable dosage delivery and convenience.
Typically, parenteral administration is the most reliable means of delivering a pharmaceutical to a patient. See, Goodman et al., Goodman and Gilman's Pharmacological Basis of Therapeutics, Pergamon Press, Elmsford, N.Y. (1990) and Pratt et al. Principles of Drug Action: The Basis of Pharmacology, Churchill Livingstone, New York, N.Y. (1990). Each parenteral mechanism insures that a prescribed dosage of the pharmaceutical agent is inserted into the fluid compartment of the body where it can be transported. The disadvantage of these modes of delivery is that they require an invasive procedure. The invasive nature of administration is inconvenient, painful and subject to infectious contamination.
Enteral and topical administration are more convenient, generally non-painful, and do not predispose to infection, however both have limited utility. The gastrointestinal and dermal surfaces present formidable barriers to transport and therefore, some pharmaceutical agents are not absorbed across these surfaces. Another drawback to patient directed modes of administration (enteral, topical and subcutaneous) is compliance. Pharmaceutical agents that have a short half-life require multiple daily doses. As the number of doses increases, patient compliance and therapeutic efficacy decrease. Simplified and/or less frequent administration schedules can aid in optimizing patient compliance. Wilson et al. (1991) Harrison's Principles of Internal Medicine, 12th Ed., McGraw-Hill, Inc., New York, N.Y.
The skin is an efficient barrier to the penetration of water soluble substances, and the rate of transdermal pharmaceutical agent absorption is primarily determined by the agent's lipid solubility, water solubility, and polarity. Highly polar or water soluble pharmaceutical agents are effectively blocked by the skin. Even very lipophilic pharmaceutical agents penetrate the dermis very slowly compared with the rate of penetration across cell membranes. See Pratt et al. supra.
Efforts to develop more effective and convenient modes of pharmaceutical administration have led to the development of transdermal delivery systems. Many current transdermal pharmaceutical agent delivery systems rely upon pharmaceutical agents that are absorbed when admixed with inert carriers. See Cooper et al. (1987) “Penetration Enhancers”, in Transdermal Delivery of Drugs, Vol. II, Kyodonieus et al., Eds., CRC Press, Boca Raton, Fla. Few pharmaceutical agents fit this profile and those which do are not always predictably absorbed. Various forms of chemical enhancers, such as those enhancing lipophilicity, have been developed to improve transdermal transport when physically mixed with certain therapeutic agents and provide more predictable absorption. See for example, U.S. Pat. Nos. 4,645,502; 4,788,062; 4,816,258; 4,900,555; 3,472,931; 4,006,218; and 5,053,227. Carriers have also been coupled to pharmaceutical agents to enhance intracellular transport. See Ames et al. (1973) Proc. Natl. Acad. Sci. USA, 70:456-458 and (1988) Proc. Int. Symp. Cont. Rel. Bioact. Mater., 15:142.
Similar to the problems inherent in trans-dermal delivery of pharmaceuticals, the blood-brain barrier is an obstacle to CNS drug delivery. In fact, the blood-brain barrier is considered to be a “bottleneck” in brain drug development, and is perhaps the single most important limitation on the future growth of neurotherapeutics. (Pardridge, W. M., The Blood-Brain Barrier: Bottleneck in Brain Drug Development, The Journal of the American Society for Experimental NeuroTherapeutics, Vol 2, 3-14, January 2005; Pardridge, W. M. Brain drug targeting: the future of brain drug development. Cambridge, UK: Cambridge University Press, 2001.) The BBB is formed by the brain capillary endothelium and prevents transport of approximately 100% of large-molecules (such as monoclonal antibodies, recombinant proteins, antisense or gene therapeutics) and more than 98% of all small-molecule drugs into the brain. Although the average molecular mass of a CNS-active drug is 357 daltons, even a small, 100 dalton molecule such as histamine does not pass through the BBB when infused into a mouse and allowed to distribute over thirty minutes time. In fact, a review of the Comprehensive Medicinal Chemistry database shows that, of more than 7000 small molecule drugs, only 5% treat the CNS, and this 5% treats only depression, schizophrenia, and insomnia.
Thus, most drugs do not cross the BBB. Unfortunately, many disorders of the central nervous system (CNS) could benefit from improved drug therapy directed towards the CNS. While there is relatively little research with respect to agents known to cross the BBB, there are characteristics that are predictive of a likelihood of success of delivery into the CNS. These are: 1) molecular mass under a 400-500 Dalton threshold, and 2) high lipid solubility. Presently, only four categories of CNS disorders respond to such molecules, including affective disorders, chronic pain, and epilepsy. Migraine headache may be considered a CNS disorder, and could also be included in this category. In contrast, patients with diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, A.L.S., multiple sclerosis, neuro-AIDS, brain cancer, stroke, brain or spinal cord trauma, autism, lysosomal storage disorders, fragile X syndrome, inherited ataxias, and blindness have very limited options with respect to pharmaceutical treatments. (There has been some success with L-DOPA treatment in Parkinson's patients, and multiple sclerosis can be treated with cytokines acting on the peripheral immune system.) (See generally, Partridge, supra).
In many of the above listed disorders, delivery across the BBB is the rate limiting problem in gene therapy or enzyme replacement therapy. Many of these disorders could be treated with drugs, enzymes or genes already discovered. However, these drugs do not cross the BBB and cannot be considered for therapeutic use for that reason. Because of the impermeability of the BBB, other approaches to drug delivery into the CNS must be used. These include the use of small molecules, trans-cranial brain drug delivery, and BBB disruption. However, none of these approaches provide solutions to the BBB problem that can be practically implemented in a large number of patients. (Pardridge, W. M., “The Blood-Brain Barrier”)
Saposins, a family of small (˜80 amino acids) heat stable glycoproteins, are essential for the in vivo hydrolytic activity of several lysosomal enzymes in the catabolic pathway of glycosphingolipids (see Grabowski, G. A., Gatt, S., and Horowitz, M. (1990) Crit. Rev. Biochem. Mol. Biol. 25, 385-414; Furst, W., and Sandhoff, K., (1992) Biochim. Biophys. Acta 1126, 1-16; Kishimoto, Y., Kiraiwa, M., and O'Brien, J. S. (1992) J. Lipid. Res. 33, 1255-1267). Four members of the saposin family, A, B, C, and D, are proteolytically hydrolyzed from a single precursor protein, prosaposin (see Fujibayashi, S., Kao, F. T., Hones, C., Morse, H., Law, M., and Wenger, D. A. (1985) Am. J. Hum. Genet. 37, 741-748; O'Brien, J. S., Kretz, K. A., Dewji, N., Wenger, D. A., Esch, F., and Fluharty, A. L. (1988) Science 241, 1098-1101; Rorman, E. G., and Grabowski, G. A. (1989) Genomics 5, 486-492; Nakano, T., Sandhoff, K., Stumper, J., Christomanou, H., and Suzuki, K. (1989) J. Biochem. (Tokyo) 105, 152-154; Reiner, O., Dagan, O., and Horowitz, M. (1989) J. Mol. Neurosci. 1, 225-233). The complete amino acid sequences for saposins A, B, C and D have been reported as well as the genomic organization and cDNA sequence of prosaposin (see Fujibayashi, S., Kao, F. T., Jones, C., Morse, H., Law, M., and Wenger, D. A. (1985) Am. J. Hum. Genet. 37, 741-748; O'Brien, J. S., Kretz, K. A., Dewji, N., Wenger, D. A., Esch, F., and Fluharty, A. L. (1988) Science 241, 1098-1101; Rorman, E. G., and Grabowski, G. A. (1989) Genomics 5, 486-492). A complete deficiency of prosaposin with mutation in the initiation codon causes the storage of multiple glycosphingolipid substrates resembling a combined lysosomal hydrolase deficiency (see Schnabel, D., Schroder, M., Furst, W., Klien, A., Hurwitz, R., Zenk, T., Weber, J., Harzer, K., Paton, B. C., Poulos, A., Suzuki, K., and Sandhoff, K. (1992) J. Biol. Chem. 267, 3312-3315).
Saposins are defined as sphingolipid activator proteins or coenzymes. Structurally, saposins A, B, C, and D have approximately 50-60% similarity including six strictly conserved cysteine residues (see Furst, W., and Sandhoff, K., (1992) Biochim. Biophys. Acta 1126, 1-16) that form three intradomain disulfide bridges whose placements are identical (see Vaccaro, A. M., Salvioli, R., Barca, A., Tatti, M., Ciaffoni, F., Maras, B., Siciliano, R., Zappacosta, F., Amoresano, A., and Pucci, P. (1995) J. Biol. Chem. 270, 9953-9960). All saposins contain one glycosylation site with conserved placement in the N-terminal sequence half, but glycosylation is not essential to their activities (see Qi. X., and Grabowski, G. A. (1998) Biochemistry 37, 11544-11554; Vaccaro, A. M., Ciaffoni, F., Tatti, M., Salvioli, R., Barca, A., Tognozzi, D., and Scerch, C. (1995) J. Biol. Chem. 270, 30576-30580). In addition, saposin A has a second glycosylation site in C-terminal half.
All saposins and saposin-like proteins and domains contain a “saposin fold” when in solution. This fold is a multiple α-helical bundle motif, characterized by a three conserved disulfide structure and several amphipathic polypeptides. Despite this shared saposin-fold structure in solution, saposins and saposin-like proteins have diverse in vivo biological functions in the enhancement of lysosomal sphingolipid (SL) and glycosphingolipid (GSL) degradation by specific hydrolases. Because of these roles, the saposins occupy a central position in the control of lysosomal sphingolipid and glycosphingolipid metabolisms (see Kishimoto, Y., Kiraiwa, M., and O'Brien, J. S. (1992) J. Lipid. Res. 33, 1255-1267; Fujibayashi, S., Kao, F. T., Hones, C., Morse, H., Law, M., and Wenger, D. A. (1985) Am. J. Hum. Genet. 37, 741-748; O'Brien, J. S., Kretz, K. A., Dewji, N., Wenger, D. A., Esch, F., and Fluharty, A. L. (1988) Science 241, 1098-1101).
The structural characteristic of these saposins is of great importance to the diverse mechanisms of activation. Since all of these proteins have high sequence similarity, but different mechanisms of action with lipid membranes, one can speculate that the specific biological functions of saposins and saposin-like proteins are the result of the differential interactions with the biological membrane environments. In vitro, saposin A enhances acid β-glucosidase activity at μM concentration, but saposin C deficiency leads to glucosylceramide storage and a “Gaucher disease-like” phenotype (see Schnable, D., Schroder, M., and Sandhoff, K. (1991) FEBS Lett. 284, 57-59; Rafi. M. A., deGala, G., Zhang, X. L., and Wenger, D. A. (1993) Somat. Cell Mol. Genet. 19, 1-7). Activation of saposin B takes place through solubilizing and presenting glycosphingolipid substrates to lysosomal enzymes (see Furst, W., and Sandhoff, K., (1992) Biochim. Biophys. Acta 1126, 1-16).
Saposin C promotes acid β-glucosidase activity by inducing in the enzyme conformational change at acidic pH (see Berent, S. L., and Radin, N. S. (1981) Biochim. Biophys. Acta 664, 572-582; Greenberg, P., Merrill, A. H., Liotta, D. C., and Grabowski, G. A. (1990) Biochim. Biophys. Acta 1039, 12-20; Qi. X., and Grabowski, G. A. (1998) Biochemistry 37, 11544-11554). This interaction of saposin C with the enzyme occurs on negatively charged phospholipid surfaces. In vitro and ex vivo saposins A and D function to enhance the degradation of galactosylceramide and ceramide/sphingomyelin, respectively (see Harzer, K., Paton, B. C., Christomanou, H., Chatelut, M., Levade, T., Hiraiwa, M. and O'Brien, J. S. (1997) FEBS Lett. 417, 270-274; Klien, A., Henseler, M., Klein, C., Suzuki, K., Harzer, K., and Sandhoff, K. (1994) Biochem. Biophys. Res. Commun, 200, 1440-1448). Patients lacking the individual saposins B and C showed a variant form of metachromatic leukodystrophy and Gaucher disease, respectively. (see Wenger, D. A., DeGala, G., Williams, C., Taylor, H. A., Stevenson, R. E., Pruitt, J. R., Miller, J., Garen, P. D., and Balentine, J. D. (1989) Am. J. Med. Genet. 33, 255-265) (see Christomanou, H., Aignesberger, A., and Linke, R. P. (1986) Biol. Chem. Hoppe-Seyler 367, 879-890).
The primary physiological function of saposin C has been defined by a glycosphingolipid (GSL) storage disease similar to neuronopathic “Gaucher's disease” in patients with a deficiency of the protein. Saposin C is a critical physiologic activator for the lysosomal enzyme, acid β-glucosidase. In addition to stimulating the glucosylceramide degradation by acid β-glucosidase, saposin C has several other potential roles. These include inter-membrane transport of gangliosides and GSLs, reorganization and destabilization of phospholipids-containing membranes, and fusion of acid phospholipids vesicles (see Hiraiwa, M., and Soeda, S. et al. (1992) Proc. Natl. Acad. Sci. USA, 89, 11254-11258; You, H. X., and Yu, L. et al., (2001) FEBS Lett. 503, 97-102; You, H. X. and Qi, X. et al. (2003) Biophys. J. 84, 2043-2057; Vaccaro, A. M., and Tatti, M. et al., (1994) FEBS Lett. 349, 181-186; Wang, Y., and Grabowski, G. et al., Biochem. Biophys., 415: 43-53; Qi, X. and Chu, Z., (2004) Arch. Biochem. Biophys., 424: 210-218). Saposin C associates with phophatidyserine (PS) membranes by embedding its amino- and carboxyl-end helices into the outer leaflet of membranes (see Qi, X. and Grabowski, G. A., (2001) J. Biol. Chem., 276, 27010-27017). Increasing evidence indicates that intereactions of saposins with appropriate membranes are crucial for their specificity and activity.
Moreover, PSAP, the precursor of saposins, is a neurotropic factor with in vitro neuritogenic, in vivo nerve growth promoting, and apoptosis protection properties (see Qi, X. and Qin, W. et al. (1996) J. Biol. Chem., 217, 6874-6880; O'Brien, J. S. and Carson, G. S. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 9593-9596; Qi, X. and Kondoh, K. et al. (1999) Biochemistry 38, 6284-6291; Kotani, Y. S, and Matsuda, S. et al. (1996) J. Neurochem. 66, 2019-2025; Koani, Y. and Matsuda, S. et al. (1996) J. Neurochem. 66, 2197-2200; Tsuboi, K. and Hiraiwa, M. et al. (1998) Brain Res. Dev. Brain Res. 110, 249-255). Such neuritogenic functions are mediated through sequences in the NH2-terminal half of saposin C (see Qi, X. and Qin, W. et al. (1996) J. Biol. Chem. 271, 6874-6880; O'Brien, J. S, and Carson, G. S. et al. (1995) FASEB J. 9, 681-685). The minimum sequence required for in vitro neuritogenic activity spans amino acid residues 22-31 of saposin C in humans and mice. Neurological functions of PSAP and saposin C are mediated by activation of the enzymes in the MAPK pathway through a G-protein-associated cell membrane receptor in a number of neuralgia-derived cells (see Campana, W. M. and Hiraiwa, M. et al. (1996) Biochem. Biophys. Res. Commun. 229, 706-712; Hiraiwa, M. and Campana, W. M. et al. (1997) Biochem. Biophys. Res. Commun. 240, 415-418).
Human and mouse PSAP genetic defects result in total saposin deficiency (see Harzer, K. and Paton, B. C. et al. (1989) Eur. J. Pediatr. 149, 31-39; Hulkova, H., and Cervenkova, M. et al. (2001) Hum. Mol. Genet. 10, 927-940; Fujita, N. and Suzuki, K. et al., Hum. Mol. Genet. 5, 711-725). This deficiency can lead to aberrant accumulation of multivesicular bodies (MVBs), as observed in the skin fibroblasts from PSAP-deficient patients (see Harzer, K. and Paton, B. C. et al. (1989) Eur. J. Pediatri. 149, 31-39; Burkhardt, J. K. and Huttler, S. et al. (1997) Eur. J. Cell Biol. 73, 10-18). Further, the sinusoidal cells in liver from a PSAP-deficient patient has been observed to be crowded with multivesicular inclusions (see Sandhoff, K. and Kolter, T. et al. (2000) The Metabolic and Molecular Bases of Inherited Disease, 3371-3388; Harzer, K. and Paton, B. C. et al. (1989) Eur. J. Pediatr. 149, 31-39). Similar MVB structures also were found in fibroblasts from a saposin C-deficient patient (see Pampols, T. and Pineda, M. et al. (1999) Acta Neuropathol. 97, 91-97). In PSAP −/− (double-knock out) mice, inclusions consisting of numerous concentric lamellar bodies and dense granular structures were noted in a variety of tissues and cells (see Oya, Y., and Nakayasu, H. et al. (1998) Acta Neuropathol 96, 29-40). Thin sections of mouse PSAP −/− cells revealed a selective accumulation of MVBs by electron microscopy (see Morales, C. R. and Zhao, Q. et al. (1999) Biocell 23, 149-160).
MVBs, a subset of the late endosomes, have a crucial role in communications by vesicular transport between the trans-Golgi network, the plasma membrane, and lysosomal/vacuolar organelles (see Katzman, D. J. and Odorizzi, G. et al. (2002) Nat. Rev. Mol. Cell. Biol. 3, 893-905). One function of MVBs is to maintain the cellular homeostasis required for neuronal development and growth. The hypothetical “signaling endosome” model explains that the ligand-receptor complex on an endosomal signaling platform is transported retrogradely from the distal axon to the cell body to promote gene expression and neuron survival (see Ginty, D. D. and Segal, R. A. (2002) Curr. Opin. Neurobiol. 12, 268-274). The abnormalities in MVB structures in neurons of PSAP−/− mice may disrupt the retrograde movement of neurotrophins via vesicular signaling transports and may impair the development of neuronal cells in the CNS.
Introducing exogenous PSAP or saposin C into the medium of cultured fibroblasts from the PSAP-deficient patient reverses the aberrant accumulation of MVBs, suggesting that saposin C is a key regulatory molecule in MVB formation (see Burkhardt, J. K. and Huttler, S. et al. (1997) Eur. J. Cell Biol. 73 10-18; Chu, Z., and Witte, D. P. et al. (2004) Exp. Cell Res.).
In addition to mediating MVB formation, saposin plays a role in membrane fusion. Membrane fusion is a major event in biological systems driving secretion, endocytosis, exocytosis, intracellular transport, fertilization, and muscle development (see Christomanou, H., Chabas, A., Pampols, T., and Guardiola, A. (1989) Klin, Wochenschr. 67, 999-1003). Recent experimental evidence generated by this inventor has indicated that saposin-lipid membrane interactions play a critical role in saposin-mediated membrane fusion of lipids thereby facilitating transport of active agents across these biological membranes.
The present invention also relates to a method of administering imaging agents across cellular membranes including the blood-brain barrier using saposin C containing liposomes. Non-invasive imaging techniques can be used to monitor the distribution and efficacy of liposomal delivery systems, thereby facilitating the evaluation and clinical application of gene therapy or therapeutic treatment using liposomes. Imaging agents may use magnetic resonance, fluorescence, or CT/PET as a means of detection. However, key obstacles to successful use of imaging agents to monitor liposome delivery are ease of detection, availability of pertinent technology and ease and efficiency of delivery.
With respect to using liposomes to deliver imaging agents, lipophilic molecules are generally appropriate, though the present invention is not limited to use with such molecules. Without intending to be limited by theory, lipophilic dyes or dyes containing a lipophilic moiety may intercalate into the liposomal membrane or reconstitute into the lipid core of liposomal structures. Examples of such dyes known in the art are the indocarbocyanine dye, DiI. DiI is a fluorescent carbocyanine dye that is routinely used to label lipid membranes. Other similar dyes are DiA or DiaO as described in Honig, M. G. et al, DiI and DiO: versatile fluorescent dyes for neuronal labeling and pathway tracing. Trends Neurosci. 12:333-335, 340-331, 1989. Other lipophilic dyes that may be used with the present invention include PKH2, NeuroVue Green, PKH 26, NeuroVue Red, and NeuroVue Maroon, as described by Fritzsch, et al. Diffusion and Imaging properties of Three New Lipophilic Tracers, Neuro Vue Maroon, Neuro Vue and Neurovue Green and their use for Double and Triple Labeling of Neuronal Profile, manuscript. Any of these dyes may be used with the present invention described herein, either alone or in combination.
Also used in the art and appropriate to the present invention are imaging agents having two or more imaging properties. Such agents allow the researcher or clinician the ability to use multiple methods of imaging to detect administered imaging agents. An example of such agents are the so-called PTIR dyes as described by Li, H., et al., MR and Fluorescent Imaging of Low-Density Lipoprotein Receptors, Acad Radiol. 2004; 11:1251-1259, incorporated herein by reference. These dyes contain both a fluorophore and a Gd(III) moiety that allow for detection via magnetic resonance imaging (MRI) or confocal fluorescence microscopy. The lipophilic side chain facilitates the intercalation of the dye into phospholipid monolayers. Thus, these dyes are appropriate for use with liposomal delivery systems such as the one described herein.
Proton MR imaging offers the advantages of being noninvasive, tomographic, and of high resolution. In recent years, magnetic resonance imaging (MRI) has emerged as a powerful tool in clinical settings because it is noninvasive and yields an accurate volume rendering of the subject. See generally, U.S. Pat. No. 6,962,686 Kayyem, et al. entitled Cell-specific gene delivery vehicles. These advantages make MRI the technique of choice in both medical imaging and as an imaging tool for use in biological experiments. Unlike light-microscope imaging techniques based upon the use of dyes or fluorochromes, MRI does not produce toxic photobleaching by-products. Furthermore, unlike light-microscopy, MRI is not limited by light scattering or other optical aberrations to cells within approximately only one hundred microns of the surface. Agents having MRI properties such as those described above may be used with the present invention.
Accordingly, there exists a significant need for nontoxic agents which can improve the delivery or transport of pharmaceutical or imaging agents across or through biological membranes, including the blood-brain barrier. The present invention fulfills these needs.